Method for Raman chemical imaging of endogenous chemicals to reveal tissue lesion boundaries in tissue

ABSTRACT

Apparatus and methods for spatially resolved Raman detection of molecules indicative of the borders of lesions with normal tissue are disclosed. A region of biological tissue was illuminated with monochromatic light. A Raman shifted light signal is detected from endogenous molecules in the region, the molecules being spatially organized in a localized first area of the region. These molecules are indicative of a border between normal tissue and a lesion. The Raman shifted light signal is then spatially resolved in at least one direction.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority pursuant to 35 U.S.C. 119(e) toU.S. Provisional Application No. 60/301,708 filed 06/28/2001 which isincorporated herein by reference in its entirety including incorporatedmaterial.

FIELD OF THE INVENTION

[0002] The field of the invention is the field of tissue evaluation and,more particularly, the field of tissue evaluation using opticaldetection of light which has been Raman shifted in frequency.

BACKGROUND OF THE INVENTION

[0003] Cancer is the second leading cause of death in the United Statesand over 1.2 million people are diagnosed with this disease annually.Cancer is significant, not only in lives lost, but also in the $107billion cost to the United States economy in 2000 according to theNational Institutes of Health. It is widely recognized among the cancerresearch community, that there is a need to develop new tools tocharacterize normal, precancerous, cancerous, and metastatic cells andtissues at a molecular level. These tools are needed to help expand ourunderstanding of the biological basis of cancers. Molecular analysis oftissue changes in cancer improve the quality and effectiveness of cancerdetection and diagnosis strategies. The knowledge gained through suchmolecular analyses helps identify new targets for therapeutic andpreventative agents.

[0004] Diagnosis of cancer is the first critical step to cancertreatment. Included in the diagnosis is the type and grade of cancer andthe stage of progression. This information drives treatment selection.When cancer is suspected, a patient will have the tumor removed orbiopsied and sent for histopathology analyses. Conventional handlinginvolves the tissue undergoing fixation, staining with dyes, mountingand then examination under a microscope for analysis. Typically, thetime taken to prepare the specimen is of the order of one day. Thepathologist will view the sample and classify the tissue as malignant orbenign based on the shape, color and other cell and tissuecharacteristics. The result of this manual analysis depends on thechoice of stain, the quality of the tissue processing and staining, andultimately on the quality of education, experience and expertise of thespecific pathologist.

[0005] Early definitive detection and classification of cancerousgrowths is often crucial to successful treatment of this disease.Currently, several biopsy techniques are used as diagnostic methodsafter cancerous lesions are identified. In the case of breast cancer,lesions are typically identified with mammography or self breast exam.The most reliable method of diagnosis is examination ofmacroscopic-sized lesions. Macroanalysis is performed in conjunctionwith microscopic evaluation of paraffin-embedded biopsied tissue whichis thin-sectioned to reveal microscale morphology.

[0006] The detection and diagnosis of cancer is typically accomplishedthrough the use of optical microscopy. A tissue biopsy is obtained froma patient and that tissue is sectioned and stained. The prepared tissueis then analyzed by a trained pathologist who can differentiate betweennormal, malignant and benign tissue based on tissue morphology. Becauseof the tissue preparation required, this process is relatively slow.Moreover, the differentiation made by the pathologist is based on subtlemorphological differences between normal, malignant and benign tissuebased on tissue morphology. For this reason, there is a need for animaging device that can rapidly and quantitively diagnose malignant andbenign tissue.

[0007] Alternatives to traditional surgical biopsy include fine needleaspiration cytology and needle biopsy. These non-surgical techniques arebecoming more prevalent as breast cancer diagnostic techniques becausethey are less invasive than biopsy techniques that harvest relativelylarge tissue masses. Fine needle aspiration cytology has the advantageof being a rapid, minimally invasive, non-surgical technique thatretrieves isolated cells that are often adequate for evaluation ofdisease state. However, in fine needle biopsies intact breast tissuemorphology is disrupted often leaving only cellular structure foranalysis which is often less revealing of disease state. In contrast,needle biopsies use a much larger gauge needle which retrieve intacttissue samples that are better suited to morphology analysis. However,needle biopsies necessitate an outpatient surgical procedure and theresulting needle core sample must be embedded or frozen prior toanalysis.

[0008] A variety of “optical biopsy” techniques have potential asnon-invasive, highly sensitive approaches that will augment, or even bealternatives to current diagnostic methods for early detection of breastcancer. “Optical biopsies” employ optical spectroscopy to non-invasivelyprobe suspect tissue regions in situ, without extensive samplepreparation. Information is provided by the resultant spectroscopicallyunique signatures that may allow differentiation of normal and abnormaltissues. Despite years of research and development, two techniques thathave not realized their potential are:

[0009] (1) fluorescence optical biopsies, which fails due to thenonspecific nature of tissue autofluorescence; and

[0010] (2) near-infrared optical diagnostics, in particular non-invasiveglucose sensing, which fails due to interference from tissue majorcomponents, including predominantly water.

[0011] In contrast to other techniques, Raman spectroscopy holds promiseas an optical biopsy technique that is anticipated to be broadlyapplicable for characterization of a variety of cancerous diseasestates. A number of researchers have shown that Raman spectroscopy ofmasses of cells has utility in differentiating normal vs. malignanttissue and differentiating normal vs. benign tissue. In general, theRaman spectra of malignant and benign tissues show an increase inprotein content and a decrease in lipid content versus normal breasttissue, demonstrating that cancer disease states impact the chemistry ofthe tissue.

[0012] However, Raman spectroscopy has not been able to differentiatebenign vs. malignant tissues due to the spectral similarities of thesetissue types. In addition, Raman spectroscopy of breast tissue samplesrequires large numbers of cell populations. If only a small portion ofthe cells are cancerous, as in the early stages of lesion development,then Raman spectroscopy of a large number of such cells will beinsensitive to the disease. It would be advantageous to have a techniquecapable of the spatial sensitivity needed for discrimination ofcancerous from normal cells in early stage breast cancer diagnosis.

[0013] Chemical imaging based on optical spectroscopy, in particularRaman spectroscopy, provides the clinician with important information.Chemical imaging simultaneously provides image information on the size,shape and distribution (the image morphology) of molecular chemicalspecies present within the sample. By utilizing molecular-specificimaging, based on chemical imaging, the trained clinician can make adetermination on the disease-state of a tissue or cellular sample basedon recognizable changes in morphology without the need for samplestaining or modification.

[0014] Apparatus for Raman Chemical Imaging (RCI) has been described bythe inventors in U.S. Pat. No. 6,002,476, and in co-pending U.S.Non-Provisional application Ser. No. 09/619,371 filed Jul. 19, 2000which claims benefit of U.S. Provisional Application Ser. No. 60/144,518filed Jul. 19, 1999. The above identified U.S. patents, patentapplications, and publications are hereby incorporated by reference.

OBJECTS OF THE INVENTION

[0015] It is an object of the invention to produce apparatus and methodsusing Raman shifted light for diagnosis of lesions in tissue. It is anobject of the invention to produce apparatus and methods for diagnosisof tissue samples excised from a patient. It is an object of theinvention to produce apparatus and methods for in vivo diagnosis oftissue. It is an object of the invention to produce apparatus andmethods for finding a lesion in vivo in tissue. It is an object of theinvention to produce apparatus and methods for determining the bordersof lesions in vivo and in tissue samples excised from a patient. It isan object of the invention to produce apparatus and methods forspatially resolving Raman shifted light from tissue in vivo and intissue samples. It is an object of the invention to produce apparatusand methods for imaging a lesion with light which has been Ramanshifted.

SUMMARY OF THE INVENTION

[0016] Raman chemical imaging is used to differentiate between normaltissue and benign and malignant lesions. In particular, Raman chemicalimaging is shown to be sensitive to calcified tissue and to carotenoidmolecules. Carotenoid molecules are concentrated at the border of alesion, and can be used to indicate the borders of the lesion. Spatiallyresolved Raman signals indicate lesions and borders of lesions. Othermolecular species which may be indicative of border regions are noted.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017]FIG. 1A. Brightfield reflectance microscope image of a calcifiedlesion from a five micron thick frozen section biopsy.

[0018]FIG. 1B. Magnified region indicated from FIG. 1A.

[0019]FIG. 1C. Raman Chemical Image (RCI) of the spatial distribution ofcalcification (calcium hydroxyapetite) and background (microscope slide)

[0020]FIG. 2A. Brightfield reflectance microscope image of a region ofcalcified lesion as indicated in FIG. 1A.

[0021]FIG. 2B. Polarized light image of the region of interest indicatedin FIG. 1A.

[0022]FIG. 2C. Raman Chemical Image (RCI) indicating calcified tissue.

[0023]FIG. 2D. Raman spectral data from two regions of interestindicated in FIG. 2C showing the different chemical composition of theseregions.

[0024]FIG. 3A. Brightfield reflectance microscope image of a 5 micronthin section of human breast tissue biopsy sample showing a lesion andthe adjacent tissue.

[0025]FIG. 3B. Magnified region of interest indicated in FIG. 3A.

[0026]FIG. 3C. Raman Chemical Image (RCI) of the endogenous caroteniod(beta-carotene) that shows the border of the lesion.

[0027]FIG. 3D. Raman spectra obtained with a tunable filter for twocircled regions indicated in FIG. 3C.

[0028]FIG. 4. Amount of carotenoid as determined from its spectralsignal along the diagonal A-A′ of the view shown in FIG. 1C.

[0029]FIG. 5. Preferred embodiment of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0030] Raman Spectroscopy

[0031] When light interacts with matter, a portion of the incidentphotons are scattered in all directions. A small fraction of thescattered radiation differs in frequency (wavelength) from theilluminating light. If the incident light is monochromatic (singlewavelength) as it is when using a laser source or other sufficientlymonochromatic light source, the scattered light which differs infrequency may be distinguished from the light scattered which has thesame frequency as the incident light. Furthermore, frequencies of thescattered light are unique to the molecular or crystal species present.This phenomenon is known as the Raman effect.

[0032] In Raman spectroscopy, energy levels of molecules are probed bymonitoring the frequency shifts present in scattered light. A typicalexperiment consists of a monochromatic source (usually a laser) that isdirected at a sample. Several phenomena then occur including Ramanscattering which is monitored using instrumentation such as aspectrometer and a charge-coupled device (CCD). Similar to an infraredspectrum, a Raman spectrum reveals the molecular composition ofmaterials, including the specific functional groups present in organicand inorganic molecules and specific vibrations in crystals. Ramanspectrum analysis is useful because each resonance exhibits acharacteristic ‘fingerprint’ spectrum, subject to various selectionrules. Peak shape, peak position and the adherence to selection rulescan also be used to determine molecular conformation information(crystalline phase, degree of order, strain, grain size, etc.). Unlikeinfrared spectroscopy, a single Raman spectrometer can be applied to themolecular characterization of organic and inorganic materialssimultaneously. Other advantages of Raman over traditional infraredspectroscopy include the ability to analyze aqueous phase materials andthe ability to analyze materials with little or no sample preparation.Deterrents to using Raman spectroscopy as opposed to infraredspectroscopy include the relatively weak nature of the Raman phenomenonand interferences due to fluorescence. In the past several years, anumber of key technologies have been introduced into wide use that haveenabled scientists to largely overcome the problems inherent to Ramanspectroscopy. These technologies include high efficiency solid statelasers, efficient laser rejection filters, and silicon charge coupleddevice (CCD) detectors.

[0033] In Raman spectroscopy instruments, a linear CCD array istypically positioned at the exit focal plane of single stage, low fnumber Raman monochromators for efficient collection of dispersive Ramanspectra. The monochromator disperses the Raman shifted light, and theCCD array typically produces a signal which is proportional to theintensity of the Raman signal vs wavelength.

[0034] Raman Chemical Imaging (RCI)

[0035] In many respects, Raman chemical imaging is an extension of Ramanspectroscopy. Raman chemical imaging combines Raman spectroscopy anddigital imaging for the molecular-specific analysis of materials. Muchof the imaging performed since the development of the first Ramanmicroprobes has involved spatial scanning of samples beneath Ramanmicroprobes in order to construct Raman “maps” of surfaces.Historically, Raman imaging systems have been built using this so calledflying spot (“point-scanning”) approach, where a laser beam is focusedto a spot and is scanned over the object field, or likewise a linescanning approach, where the laser spot is broadened in one directionby, for example, a cylindrical lens, and the two dimensional imageformed on a CCD array has one spatial dimension and one wavelengthdimension. Raman chemical imaging techniques have only recently achieveda degree of technological maturity that allows the simultaneouscollection of high-resolution (spectral and spatial) data. Advancementsin imaging spectrometer technology and their incorporation intomicroscopes that employ CCDs, holographic optics, lasers, and fiberoptics have allowed Raman chemical imaging to become a practicaltechnique for material analysis.

[0036] Imaging spectrometers include Fabry Perot angle rotated or cavitytuned liquid crystal (LC) dielectric filters, acousto-optic tunablefilters, and other LC tunable filters (LCTF) such as Lyot Filters andvariants of Lyot filters such as Solc filters and the most preferredfilter, an Evan's split element liquid crystal tunable filter, which isdescribed in the March (1999) issue of Analytical Chemistry on page175A. Other preferred wavelength filtering means comprisepolarization-independent imaging interferometers such as Michelson,Sagnac, Twynam-Green, and Mach-Zehnder interferometers.

[0037] References describing the above identified techniques that can beused to obtain chemical images include:

[0038] Fiber Array Filters (FAST)—M. P. Nelson, M. L. Myrick, Appl.Spectroscopy 53, 751-759, (1999);

[0039] Dielectric Interference filters—D Batchelder, C Cheng, W Muller,B Smith, Makromol Chem Macromol. Symp 46, 171, (1991);

[0040] AOTF—P. J. Treado, I. W. Levin, E. N. Lewis, Appl. Spectrosc. 46,1211-1216, (1992);

[0041] Lyot—B. Lyot, C. R. Acad. Sci. 197:1593. (1933);

[0042] Fabry Perot—K. A. Christainsen, N. L. Bradley, M. D. Morris, R.V. Morrison, Appl. Spectrosc. 49, 120-1125, (1995);

[0043] Solc filter—A. Yariv & P. Yeh, Optical Waves in Crystals, (WileyN.Y., 1984);

[0044] Michelson Interferometer—Sybil P. Parker, Optics Source Book,(McGraw-Hill, N.Y., 1988 p143);

[0045] Sagnac Interferometer—S. Spielman, K. Fesler, C. B. Eom, T. H.Geballe, M. Fejer and A Kapitulnik, Phys. Rev. Lett., 65, 123 (1990);and

[0046] Twyman-Green Interferometer—M. Born and E. Wolf, Principles ofOptics: Electromagnetic Theory of Propogation of Light, 6th Ed,(Pergamon Press, Oxford, 1980) pp 302-305.

[0047] Mach-Zehnder—James D. Ingle, Jr., and Stanley R Crouch,Spectrochemical Analysis, (Prentice Hall, Engelwood, N.J., 1988), p 83.

[0048] Raman chemical imaging is a versatile technique that is wellsuited to the analysis of complex heterogeneous materials. In a typicalRaman chemical imaging experiment, a specimen is illuminated withmonochromatic light, and the Raman scattered light is filtered by animaging spectrometer which passes only a single wavelength range. TheRaman scattered light may then be used to form an image of the specimen.A spectrum is generated corresponding to millions of spatial locationsat the sample surface by tuning an imaging spectrometer over a range ofwavelengths and collecting images intermittently. Changing the selectedpassband (wavelength) of the imaging spectrometer to another appropriatewavelength causes a different material to become visible. A series ofsuch images can then uniquely identify constituent materials, andcomputer analysis of the image is used to produce a composite imagehighlighting the information desired. Although Raman chemical imaging ispredominately a surface technique, depth-related information can also beobtained by using different excitation wavelengths or by capturingchemical images at incremental planes of focus. Contrast is generated inthe images based on the relative amounts of Raman scatter or otheroptical phenomena such as luminescence that is generated by thedifferent species located throughout the sample. Since a spectrum isgenerated for each pixel location, chemometric analysis tools such ascorrelation analysis, Principle Component Analysis (PCA) and factorrotation, including Multivariate Curve Resolution (MCR) can be appliedto the image data to extract pertinent information otherwise missed byordinary univariate measures. A spatial resolving power of approximately250 nm has been demonstrated for Raman chemical imaging using visiblelaser wavelengths. This is almost two orders of magnitude better thaninfrared imaging which is typically limited to 20 microns due todiffraction. In addition, image definition (based on the total number ofimaging pixels) can be very high for Raman chemical imaging because ofthe use of high pixel density detectors (often 1 million plus detectorelements).

[0049] Applications of Raman chemical imaging range from the analysis ofpolymer blends, defect status analysis in semiconductor materials,inclusions in human breast tissue and the characterization of corrosionsamples. RCI provides a potential solution for obtaining bothqualitative and quantitative image information about molecularcomposition and morphology of breast lesions allowing a more accuratemedical diagnosis than traditional imaging methods.

[0050] Breast Tissue Results

[0051] Raman spectra can potentially reveal a wealth of informationabout molecular properties of tissues. RCI compounds this information byallowing variations in these properties throughout the tissue to beprobed. FIG. 1 shows RCI data on a calcified lesion. The tissue wasexcised from the patient, and frozen. A five micron thick section wassliced from the tissue and prepared on a microscope slide for imaging ina microscope. FIG. 1A shows a brightfield reflectance image of a portionof the frozen sectioned biopsy which is then magnified in FIG. 1B. Thebrightfield image reveals light and dark regions resulting fromdifferences in refractive indices. These images, however, provide noinsight into the molecular makeup of the tissues at hand. A Ramanchemical image is shown in FIG. 1C and reveals the distribution ofcalcium hydroxyapatite based on its Raman response. FIG. 2A and FIG. 2Bshow dramatic differences in the optical microscopic image that dependon the polarization of the light. However, the Raman chemical image inFIG. 2C is unique in that it is derived from the distinct spectral Ramanshown in FIG. 2D. The Raman spectra in FIG. 2D shows the spectral“fingerprints” associated with the calcium hydroxyapatite and thebackground, (the glass microscope slide) respectively. Such Ramanspectra are the basis that allow a Raman Chemical image to be created.This ability to characterize calcifications is a critical issue in thediagnosis of breast carcinoma as calcification is a major element inmammographic evaluation and early cancer detection, and is critical forthe diagnositic pathologist to identify. The Raman spectrum of calciumsalts and protein calcium complexes is an incompletely explored area, inlarge part because of the previous unavailability of instrumentationcapable of simultaneous high resolution spatial imaging and highwavelength resolution Raman spectrochemical analyses.

[0052] Difficulties exist when trying to use non imaging Ramanspectroscopy alone to differentiate benign vs. malignant tissues due tothe spectral similarities of these tissue types and to the spectrum ofbreast conditions that may mimic cancer. In addition, non imaging Ramanspectroscopy of breast tissue samples large numbers of cell populations.If only a small portion of the cells are cancerous, as in the earlystages of lesion development, then non-imaging Raman spectroscopy willbe insensitive to the disease. It is very advantageous to have atechnique capable of the spatial imaging sensitivity needed fordiscrimination of cancerous from normal cells in early stage breastcancer diagnosis.

[0053] We have developed an imaging optical biopsy approach based onRaman chemical imaging. In comparison with non-imaging Ramanspectroscopy, our approach has the advantage that we efficiently collectspatial resolved Raman spectra so that morphometric analysis(characterization by size and shape) can be performed in conjunctionwith Raman spectral analysis. The additional morphology information isanticipated to add a critical component to the analysis of diseasestates, in part because it builds upon traditional cancer histopathologymethods and could therefore be readily adopted by pathologists. FIG. 3Ashows a brightfield image of a 5 □m thin section human breast tissuebiopsy sample viewed under the microscope. An enlarged section of thelesion is indicated and magnified in FIG. 3B to show the border orinterface between a tumor and normal tissue, where both cancerous andnormal cells are visible. The Raman chemical image of a carotenoidmolecule, β-carotene, shown in FIG. 3C reveals the location of the tumorand carotenoid molecules. Note that the carotenoid molecules areassociated with the border between the lesion and the normal tissue. TheLCTF-generated Raman spectra in FIG. 3D shows the spectral“fingerprints” associated with the tumor and the typical normal tissue,respectively. The ability to see this boundary with an inherent chemicalwithin human tissue is a unique finding with potential biological andclinical significance relating to the objective screening andcharacterization of tumor margins.

[0054]FIG. 4 shows the results of a scan of the carotenoid signal alongthe diagonal A-A′, ie along a line perpendicular to the tumor normaltissue boundary of FIG. 3C.

[0055] It is very important to know where the tumor margins are, and toknow if the tumor has infiltrated beyond the a well defined boundary andinto normal tissue. Detection of molecules indicative of the boundary isof great importance. The nutritional literature supports the idea thatcarotenoids are protective from cancer. It is surmised but not proven bythe inventors that such protective molecules accumulate in the borderregion between a lesion and normal tissue, and act to prevent the lesionfrom growing. Other molecules suggested by the nutritional literature inrelation to breast cancer are indoles, sulforaphanes, and flavonoids.Proteoglycans molecules have been noted to be associated with prostatecancer. With the Raman chemical imaging, the position of thesemolecules, and molecules which will be identified in the future, may beclearly imaged and used to show the extent and the stage of growth ofthe cancer or other lesion.

[0056] The cancerous cells shown in the lesion in FIG. 3B and 3C arealso differentiated from adjacent cells in the Raman image based onmolecular compositional variations (lipid vs. protein content primarily)and can also be used to create a Raman image of the diseased tissue. Asa result, the images are molecule-specific and more specific than imagesderived from stains. Because the Raman scattering of the tissues isintrinsic to the tissues, stains are not required and the technique issuitable for in vivo use. The Raman images are collected in only severalseconds using laser power density that does not modify the tissuesamples.

[0057] An in vivo embodiment of the invention for examining a breast 50or other non-arterial soft tissue for a lesion 51 is shown in FIG. 5. Anendoscope or other instrument 52 is used to introduce light carried byan optical fiber 53 from a monochromatic light source 54. A dichroicmirror 55 and lens 56 are shown schematically for introducing the lightinto the fiber 53. Raman light from the breast is carried from thebreast tissue back through the lens 56 and mirror 55, through a filter57 to a detector 58. The signal from the detector 58 is analyzed by acomputer system 59 and displayed on a monitor 60.

[0058] Filter 57 is most preferably a Evan's split element liquidcrystal tunable filter, which is controlled by computer 59.

[0059] The endoscope 52 is preferably an imaging endoscope orfiberscope, where light is conducted from the breast tissue to thedetector 58 in a coherent manner through a large plurality of opticalfibers. A series of two dimensional images is preferably taken as afunction of depth into the tissue and of the Raman shifted wavelength.

[0060] Results of a preferable embodiment of the invention is shown byan insert in FIG. 5, where the signal shown is a signal of a moleculeindicative of a border region between the breast 50 or other nonarterial soft tissue and the lesion 51. The spatially resolved signal ofcalcified tissue or of, for example, carotenoid molecules, is shown inthe insert as a function of depth into the breast as the needle carryingthe optical fiber is moved into the breast. The signal is showndisplayed on the display device 60. In this embodiment, a much finerneedle is used than the needle carrying an imaging endoscope. In thefine needle embodiment, the location of the lesion may be moreaccurately determined, so that fine needle aspiration cytology and/orneedle core biopsy may be performed. In the fine needle embodiment, thefilter 57 may be a normal spectrometer or a liquid crystal tunablefilter, preferably of the Evan's split element type.

[0061] Raman chemical imaging also has demonstrated utility for thequantitative assessment of lesions in breast tissues. However, there isa need to make systematic strides in the development of a RCI opticalbiopsy. RCI of animal breast tissue models have been analyzed, as wellas studies of human cancer lesions. Other lesions besides benign andmalignant tumors, such as pockets of infection and inflamation will showup in the Raman chemical images. Several data treatment methods havebeen utilized to analyze the Raman image data which include bandratioing, band shift analysis, and classical linear least squaresanalysis. Comparisons have been made between the various processingapproaches that address the utility of RCI for breast tissue componentdiscrimination.

[0062] Applications

[0063] There is a great need for an instrument that can provide: realtime detection with accuracy, decreased patient discomfort and recovery,minimal cosmetic defect of the breast, minimal distortion of the breasttissue that might make interpretation of future mammograms difficult andmost importantly provide the patient with rapid feedback on hercondition.

[0064] The user base for an instrument suitable for objective assessmentof breast lesions of will consist of medical research laboratories,University and non-affiliated hospitals, and private clinics.

[0065] On another level, the customer or end-user is the patient thatrequires the procedure be completed to determine the disease state ofher breast tissues. At this level the numbers are as follows: more than1,000,000 biopsies were conducted in 1997; the growth rate for biopsiesis almost 20% annually as clinicians struggle with how to determine thedisease state of tissue early enough to prevent radical measures; thetypical “customer” is a woman over the age of 40 that should be havingannual breast exams by a clinician; and the number of potentialcustomers is approximately 57 million (women between ages of 40 and 85).

[0066] The benefits to the target users of RCI systems will besubstantial. Configured in an endoscopic version of the technology, RCIcan be employed for “real-time” breast tissue evaluation tool that iscompatible with and complementary to existing, mature clinicalapproaches (namely, needle core biopsies). When performed in combinationthe effectiveness of breast cancer diagnosis will likely be enhanced.Benefits will include, but are not limited to, the following:

[0067] Real-time evaluation of suspicious lesions sites identifiedthrough self-breast exam and/or mammography that are made accessible vianeedle core biopsy.

[0068] Immediate feedback to the clinician as to the severity of theclinical situation. Results can be communicated to the patient by thephysician shortly after completion of Raman biopsy.

[0069] Potential information on prognostic indicators of disease such asgrowth rate through quantitative evaluation of cellular nucleic acidcomposition and proliferation associated peptides.

[0070] Minimal patient discomfort.

[0071] Minimal to no cosmetic defect of the breast.

[0072] Reduced exposure to ionizing radiation (x-rays).

[0073] Specific applications of a RCI system for evaluating breastlesions will include the following:

[0074] Discrimination of malignant vs. benign tumors

[0075] Spatial distribution of carotenoids in tissues

[0076] Spatial distribution of calcified tissue

[0077] Spatial distribution of proteins, lipids and carbohydrates intissues

[0078] Advantages Over Currently Available Technology

[0079] Traditional approaches to identification of breast lesionsinclude self-breast exam and x-ray mammography. These techniques areeffective as initial screening techniques, especially when performed incombination. Unfortunately, mammography is associated with a high falsepositive rate, resulting in 3-7 patients being biopsied for everypatient cancer diagnosed. Although many mammographic abnormalities aredefinitely benign, and others are obviously malignant, there are manylesions in which the diagnosis cannot be made with certainty based onthe mammographic appearance alone. To verify the disease-state of adetected lesion, tissue must be sampled for pathologic examination. Thismay be done with fine needle aspirates, core biopsies, or excisionalbiopsies. These samples are then prepared, stained, and inspected by atrained pathologist. This process can take several days to completebefore the patient is informed of the outcome. Raman chemical imagingtechnology has the potential to assist diagnosis of the disease state ofbreast lesions in real-time.

[0080] Currently, several biopsy techniques are used as diagnosticmethods after breast lumps are identified, typically with mammography,ultrasound, or breast examination. The most reliable method of diagnosisis examination of macroscopic-sized lesions. Macroanalysis is performedin conjunction with microscopic evaluation of paraffin-embedded biopsiedtissue which is thin-sectioned to reveal microscale morphology.

[0081] Alternatives to traditional surgical biopsy include fine needleaspiration cytology and needle core biopsy. These non-surgicaltechniques are becoming more prevalent as breast cancer diagnostictechniques because they are less invasive than conventional biopsytechniques that involve surgical incision. Fine needle aspirationcytology has the advantage of being a rapid, minimally invasive,non-surgical technique that retrieves cytologic material that is oftenadequate for evaluation of disease state. However, in fine needlebiopsies breast tissue histologic features are minimal, leaving onlycytologic features for analysis of disease state. In contrast, needlebiopsies use a much larger gauge needle which retrieve tissue samplesthat are better suited to morphology analysis. However, needle biopsiesnecessitate an outpatient surgical procedure and the resulting needlecore sample must be fixed, embedded and processed prior to analysis.

[0082] State-of-the-Art Raman Chemical Imaging Techniques

[0083] Several Raman chemical imaging technologies have evolved thatcompete with widefield tunable filter-based RCI. These techniquesinclude point scanning RCI, line imaging RCI, RCI using interferencefilters, Fourier-transform interferometry, Hadamard-transform scanningand FAST technology.

[0084] Point scanning involves taking a complete spectrum for a singleX,Y position of a sample followed by raster-scanning the sample for theremaining X,Y positions. This method offers advantages of high spectralresolution and full spectral resolution, but lacks high image definitioncapabilities and is extremely time consuming. Line imaging involvescollecting data from vertical sections of the sample characterized by asingle value of X and all values of Y, followed by subsequent scanningin the X direction. This method has the nearly the same advantages anddisadvantages as the point scanning approach, but can be done morerapidly. Field curvature artifacts are a consequence of line imagingwhich degrade image quality. The use of single or multiple interferencefilters can be used to produce a wavelength specific image(s). Thismethod is rapid, cheap and produces high definition images, but lacksspectral resolution and is susceptible to image artifacts.Fourier-transform interferometers use a mechanically driveninterferometer with a CCD-based detection system. Interferograms areimaged with the CCD for subsequent spectral interpretation for each stepof the interferometer. This method boasts good spatial resolution butsuffers from poor spectral resolution (˜100 cm⁻¹). Hadamard transformchemical imaging techniques couple Hadamard mask spatial multiplexingwith CCD-based detection to obtain two spatial and one spectraldimension of data. This method offers S/N advantages for low-light levelapplications such as Raman spectroscopy in addition to sub-nanometerspectral resolution. However, the technique suffers from fair spatialresolution and poor temporal resolution since the latter involvesscanning through numerous coding masks. Fiber array spectral translators(FAST) use a two dimensional arrangement of Raman collection fiberswhich are drawn into a one dimensional distal array at the opposite end.The one dimensional fiber stack is coupled to an imaging spectrograph.Software then extracts the spectral/spatial information which isembedded in a single CCD image frame. FAST is capable of acquiringthousands of full spectral range, position-specific Raman spectra andwavenumber-specific Raman chemical images in seconds. However, the imagedefinition of FAST is limited by the number of pixels in any onedirection of the CCD chip used in the detection system (typically nobetter than 45×45 (˜2048) imaging elements).

[0085] The ideal chemical imaging system for characterization wouldprovide fast acquisition times (seconds), high spatial resolution(sub-micron) and good spectral resolution (<0.2 nm). To date, systemsequipped with liquid crystal tunable filters are the only RCI systemthat meets these requirements.

[0086] Other Spectroscopy-Based Imaging Methods

[0087] Spectroscopic technologies that compete with Raman such asfluorescence and infrared (IR) spectroscopy are not of great concernbased on the resolution needed to see molecules on the order of 250microns. Although fluorescence has showed some promise, it suffers fromlow specificity without the use of invasive dyes or stains that requireFDA approval. IR spectroscopy cannot compete due to the difficulty withwater absorption in the IR. Tissues do not image well because of theiraqueous nature. Systems equipped with LCTFs surpass any dispersivegrating or acousto-optic tunable filter (AOTF) technology on the market.The spectral bandpass capability of the LCTF is 8 cm⁻¹ allowing for themost effective means to obtain image detail.

[0088] Traditional Biomedical Imaging Methods

[0089] Traditionally, biomedical imaging has been divided into capturingimages of live tissue (in vivo) at relatively low resolution (from 10 to1000 microns) and capturing images of excised tissue at high resolution.In vivo imaging is usually performed using non-optical modalities suchas magnetic resonance imaging, ultrasound, or x-ray tomography, whichassess the general shape and appearance of tissue in its native state;however, this approach does not provide the cellular resolutionnecessary to analyze cell types and tissue morphology. To image tissueat high resolution using conventional optical or electron microscopes,one had to slice the tissue into thin sections, otherwise the tissueabove and below the layer of interest will produce out-of-focusreflections that seriously degrade image contrast. Confocal techniquesaddress this to some extent. Excising, fixing and staining thin tissuesections is however static, is time-consuming and by the very nature ofthe process involves tissues which have been rendered non viable.

[0090] A RCI system will produce quantitative digital images of thelesion tissue that will be recognizable to the clinician who makesdisease-state determinations, in large part, based on the visualappearance of images. The appearance of suspect tissue, when viewed bythe naked eye if lesions are large enough, or via x-ray mammography, orvia magnetic resonance imaging (MRI) provides important clues to thestate of the tissue. After years of training, clinicians can basediagnosis on these subtle visual clues. Despite the best efforts ofhighly skilled professionals, early stage disease-state determination isa difficult problem. By aiding the pathologist with an image that mapsthe distribution of certain molecular species, the large number ofsubjective determinations of disease state in breast tissue biopsies canbe greatly reduced.

[0091] Although we have described certain present preferred embodimentsof our method for objective evaluation of breast tissue using Ramanimaging spectroscopy, it should be distinctly understood that ourinvention is not limited thereto, but may include equivalent methods. Itis further to be distinctly understood that the present invention is notlimited to the evaluation of breast tissue and applies to the evaluationof all tissue. Obviously, many modifications and variations of thepresent invention are possible in light of the above teachings. It istherefore to be understood that, within the scope of the appendedclaims, the invention may be practiced otherwise than as specificallydescribed. Publications, patents, and patent applications noted hereinare hereby included by reference.

We claim:
 1. A method, comprising: a) illuminating a region ofbiological tissue with monochromatic light; then b) detecting a Ramanshifted light signal from endogenous molecules in the region, themolecules spatially organized in a localized first area of the region,the molecules indicative of a border between normal tissue and a lesion;and c) spatially resolving the Raman shifted light signal in at leastone direction.
 2. The method of claim 1, where the region is imaged. 3.The method of claim 2, where the Raman shifted light from the regionpasses through a FAST fiber array spectral translator.
 4. The method ofclaim 2, where the Raman shifted light from the region passes through aFabry Perot tunable filter.
 5. The method of claim 2, where the Ramanshifted light from the region passes through an acousto-optic tunablefilter.
 6. The method of claim 2, where the Raman shifted light from theregion passes through a liquid crystal tunable filter.
 7. The method ofclaim 6, where the Raman shifted light from the region passes through aLyot filter.
 8. The method of claim 7, where the Raman shifted lightfrom the region passes through an Evan's split element liquid crystaltunable filter.
 9. The method of claim 7, where the Raman shifted lightfrom the region passes through a Solc filter.
 10. The method of claim 2,where the Raman shifted light from the region passes through apolarization-independent imaging interferometer.
 11. The method of claim10, where the Raman shifted light from the region passes through aMichelson interferometer.
 12. The method of claim 10, where the Ramanshifted light from the region passes through a Sagnac interferometer.13. The method of claim 10, where the Raman shifted light from theregion passes through a Twynam-Green interferometer.
 14. The method ofclaim 10, where the Raman shifted light from the region passes through aMach-Zehnder interferometer.
 15. The method of claim 2, where themolecules are chosen from the group consisting of indoles,sulforaphanes, carotenoids, proteoglycans, and flavonoids.
 16. Themethod of claim 15, where the molecules are carotenoid molecules. 17.The method of claim 15, where the molecules are indole molecules. 18.The method of claim 15, where the molecules are sulforphane molecules.19. The method of claim 15, where the molecules are flavanoid molecules.20. The method of claim 15, where the molecules are proteoglycanmolecules.
 21. The method of claim 2, where the region is prepared forillumination, in a step previous to step a), by excision of the tissueand by placing a specimen prepared from the tissue in position forillumination and imaging, wherein the tissue is not treated withstaining agents.
 22. The method of claim 2, where the region is preparedfor illumination, in a step previous to step a), by excision of thetissue and by placing a specimen prepared from the tissue in positionfor illumination and imaging, wherein the tissue is treated withstaining agents.
 23. The method of claim 1, where an endoscope isintroduced into the region, and the Raman shifted light signal from themolecules is detected and spatially resolved.
 24. The method of claim23, where the region is imaged using the Raman shifted light.
 25. Themethod of claim 24, where the Raman shifted light from the region passesthrough a FAST fiber array spectral translator.
 26. The method of claim24, where the Raman shifted light from the region passes through a FabryPerot tunable filter.
 27. The method of claim 24, where the Ramanshifted light from the region passes through an acousto-optic tunablefilter.
 28. The method of claim 24, where the Raman shifted light fromthe region passes through a liquid crystal tunable filter.
 29. Themethod of claim 28, where the Raman shifted light from the region passesthrough a Lyot filter.
 30. The method of claim 29, where the Ramanshifted light from the region passes through an Evan's split elementliquid crystal tunable filter.
 31. The method of claim 24, where theRaman shifted light from the region passes through apolarization-independent imaging interferometer.
 32. The method of claim31, where the Raman shifted light from the region passes through aMichelson interferometer.
 33. The method of claim 31, where the Ramanshifted light from the region passes through a Sagnac interferometer.34. The method of claim 31, where the Raman shifted light from theregion passes through a Twynam-Green Interferometer.
 35. The method ofclaim 31, where the Raman shifted light from the region passes through aMach-Zehnder Interferometer.
 36. The method of claim 23, where themolecules are chosen from the group consisting of indoles,sulforaphanes, carotenoids, proteoglycans and flavonoids.
 37. The methodof claim 36, where the molecules are carotenoid molecules.
 38. Themethod of claim 23, where a non-imaging endoscope is moved through thetissue in vivo, and the Raman shifted light signal is spatially resolvedas the endoscope is moved through the tissue.
 39. The method of claim23, where the Raman shifted light from the region passes through anEvan's split element liquid crystal tunable filter.
 40. The method ofclaim 1, further comprising; d) removing biopsy tissue material from asecond area indicated by the first area.
 41. The method of claim 40,further comprising; e) examining the biopsy tissue material for signs ofcancer.
 42. The method of claim 41, further comprising; f) excisingtissue material from a third area indicated by the first area.